WO2004105791A2 - Vaccins anti-lhrh de recombinaison - Google Patents
Vaccins anti-lhrh de recombinaison Download PDFInfo
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- WO2004105791A2 WO2004105791A2 PCT/IN2004/000153 IN2004000153W WO2004105791A2 WO 2004105791 A2 WO2004105791 A2 WO 2004105791A2 IN 2004000153 W IN2004000153 W IN 2004000153W WO 2004105791 A2 WO2004105791 A2 WO 2004105791A2
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- vaccine
- lhrh
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
- A61K39/001144—Hormones, e.g. calcitonin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0006—Contraceptive vaccins; Vaccines against sex hormones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/80—Vaccine for a specifically defined cancer
- A61K2039/812—Breast
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/80—Vaccine for a specifically defined cancer
- A61K2039/884—Vaccine for a specifically defined cancer prostate
Definitions
- the present invention relates to oligonucleotide sequences of SEQ ID Nos. 1 and 2, and polypeptide sequences of SEQ ID No. 3 and 4, useful for preparing recombinant anti-LHRH DNA and proteinic vaccines; also, a method of obtaining the vaccines; further, a vaccine compositions comprising DNA and/or proteinic vaccines optionally along with an adjuvant and/or delivery system; and lastly, a method of reducing levels of sex steroid hormones useful for controlling and/ or treating the sex-steroid hormone dependent benign prostate hypertrophy and/or prostate cancer, breast cancer, estrus of companion animals and meat quality of male animals.
- LHRH Luteinizing hormone releasing hormone
- Injecting a vaccine to generate antibodies reactive with mammalian hormone LHRH or GnRH has potential for the immuno-therapy of prostate cancer as well as for atrophy of the Injecting a vaccine to generate antibodies reactive with mammalian hormone LHRH or GnRH, has potential for the immuno-therapy of prostate cancer as well as for atrophy of the benign prostate enlargement.
- the hormone (LHRH/GnRH) is largely produced by hypothalamus in the brain, from where it travels through blood circulation to pituitary, an endocrine gland situated at the base of skull.
- the pituitary Under the influence of LHRH/GnRH, the pituitary produces and secretes two gonadotropins - FSH and LH, which in turn act on the testes in males, and ovaries in females to generate sperm or eggs, respectively. Concomitantly the hormonal cascade also causes the production of sex steroid, hormones: testosterone in males, and estrogen and progesterone in the females. These sex steroids provide an important stimulus to the accessory reproductive organs. In males, androgens promote the growth and sustain the size of the prostate, besides other accessory organs. Blocking of LHRH results in blocking of production of sperms and testosterone acting on accessory reproductive organs, such as prostate. In females, blocking of LHRH blocks ovulation and production of female sex hormones such as estrogens which promote the growth of hormone dependent breast cancers.
- LHRH The companion animals like dogs and cats, also secrete LHRH. Blocking LHRH by the bioeffective antibodies, would provide reversible control of fertility and hormone dependent manifestations such as estrus of female companion animals (Talwar et al., 1985). Immunization of male animals against LHRH will, also have application in blocking the testosterone production thereby in improving the quality of meat in animals raised for this purpose like pigs, rams, etc.
- LHRH or GnRH is common to both males and females and interestingly its molecular structure is conserved and nearly identical in animals and humans.
- rat offers a model for evaluation of the efficacy and safety of any product designed against LHRH for eventual application in humans.
- This axiom has been proven by our prior work on a semi-synthetic vaccine against LHRH (Talwar et al., 1992), which caused atrophy of the prostate in rats (Jayashankar et al, 1989) and monkeys (Giri et al., 1991). It inhibited the growth of the Dunning prostatic tumors implanted in rats (Fuerst et al., 1997).
- the present vaccine which is hereby communicated, is designed by recombinant DNA technology to enable large-scale production at low costs. It would be free of inconsistencies of carrier linkage, and avoid carrier induced epitopic suppression.
- the main object of the present invention is to design oligonucleotide sequences useful in preparing recombinant anti-LHRH DNA vaccines.
- Still another object of the present invention is to develop a vaccine composition using purified recombinant anti-LHRH protein optionally along with an adjuvant and/or a delivery system.
- Still another object of the present invention is to develop a vaccine to decrease the levels of sex-steroid hormones.
- Still another object of the present invention is to develop a vaccine to decrease the levels of sex-steroid hormone testosterone.
- Still another object of the present invention is to treat sex-steroid hormone dependent cancers.
- Still another object of the present invention is to bring about atrophy of the enlarged prostate. Still another object of the present invention is to improve the quality of the meat in male animals.
- Still another object of the present invention is to treat sex hormone dependent breast cancer.
- Still another object of the present invention is to regulate estrus of companion animals, such as dogs.
- the present invention relates to oligonucleotide sequences of SEQ ID Nos. 1 and 2, and polypeptide sequences of SEQ ID No. 3 and 4, useful for preparing recombinant anti-LHRH
- DNA and proteinic vaccines also, a method of obtaining the vaccines using oligonucleotide sequence of SEQ ID No. 1 or 2; further, the vaccine compositions comprising DNA and/or proteinic vaccines optionally along with an adjuvant and/or delivery system; and lastly, a method of reducing levels of sex steroid hormones by eliciting an effective antibody response against LHRH in mammals of both sexes using the vaccine compositions, for controlling and/ or treating the sex-steroid hormone dependent benign prostate hypertrophy and/or prostate cancer, breast cancer, estrus of companion animals, and meat quality of male animals.
- the present invention relates to oligonucleotide sequences of SEQ ID Nos. 1 and 2, and polypeptide sequences of SEQ ID No. 3 and 4, useful for preparing recombinant anti- LHRH DNA and proteinic vaccines; also, a method of obtaining the vaccines using oligonucleotide sequence of SEQ ID No.
- the vaccine compositions comprising DNA and/or proteinic vaccines optionally along with an adjuvant and/or delivery system; and lastly, a method of reducing levels of sex steroid hormones by eliciting an effective antibody response against LHRH in mammals of both sexes using the vaccine compositions, for controlling and/ or treating the sex-steroid hormone dependent benign prostate hypertrophy and/or prostate cancer, breast cancer, estrus of companion animals, and meat quality of male animals.
- SEQ ID No. 1 SEQ ID No. 1
- No.1 useful for preparing a recombinant anti-LHRH DNA vaccine.
- an oligonucleotide sequence as claimed in claim 1 wherein the said SEQ ID No.1 expressing corresponding polypeptide sequence of SEQ ID No.3 useful as a recombinant anti-LHRH proteinic vaccine.
- an oligonucleotide sequence as claimed in claim 4 wherein the said SEQ ID No.2 expressing corresponding polypeptide sequence of SEQ ID No.4 useful as a recombinant anti-LHRH proteinic vaccine.
- a method of obtaining purified recombinant anti-LHRH DNA vaccine using oligonucleotide sequence of SEQ ID No.1 or 2 comprising the steps of: ⁇ • designing an oligonucleotide sequence of SEQ ID No.1 or 2, • inserting the said sequence into a eukaryotic expression vector comprising an appropriate promoter,
- the promoter is selected from a group comprising SV40, CMV, HSV, RSV, and MMTV.
- the bacterial host is selected from a group comprising E. coli DH1, DH5alpha, C600, and XLl-Blue.
- the vectors is selected from a group comprising VR1012, VR1020, pRc/CMV, pUMVC7 and pNAC
- a method of obtaining recombinant anti-LHRH proteinic vaccine using polypeptide of SEQ ID No. 3 or 4 said method comprising the steps of:
- the promoter is selected from a group comprising T7, T5, tac, lac, pH, and ⁇ P L .
- the bacterial host is selected from a group comprising E. coli strains of BL21, BL21(DE3), BL21(DE3)pLys, TGI, and
- the vectors are selected from a group comprising pRSET, pET, pQE, and pSE420.
- a vaccine composition comprising plasmid DNA containing oligonucleotide sequence of SEQ ID No. 1 optionally along with an adjuvant and/or delivery system useful as a recombinant anti-LHRH DNA vaccine.
- a vaccine composition comprising protein of polypeptide sequence of SEQ ID No. 3 optionally along with an adjuvant and/or delivery system useful as a recombinant anti-LHRH proteinic vaccine.
- a vaccine composition comprising plasmid DNA containing oligonucleotide sequence of SEQ ID No. 2 optionally along with an adjuvant and/or delivery system useful as a useful as a recombinant anti-LHRH DNA vaccine.
- a vaccine composition comprising protein of polypeptide sequence of SEQ ID No. 4 optionally along with an adjuvant and/or delivery system useful as a recombinant anti-LHRH proteinic vaccine.
- LHRH Luteinizing hormone Releasing Hormone
- the delivery system is selected from a group comprising Microparticles of poly lactide, poly glycolide or poly-lactic-co- glycolic acid (PLGA) copolymer, Nano particles of calcium phosphate, Cationic transfection lipids, and Gene gun.
- PLGA poly-lactic-co- glycolic acid
- the sex hormones to be reduced are, testosterone in males, and progesterone and estrogen in females.
- the said method is used for controlling and/ or treating the sex-steroid hormone dependent cancers.
- said method brings down the levels of testosterone to castration levels without surgical removal of testes.
- parenteral route comprises intramuscular, intradermal, subcutaneous, intranasal, and oral route.
- multiple injections of the vaccine can be given over an extended periods of time.
- dosage of both DNA and proteinic vaccine is ranging between 20 to 500 ⁇ g per dose once or twice in a month.
- the adjuvants are selected from a group comprising alum, sodium phthalyl lipopolysaccharide (SPLPS), Freund's complete adjuvant/Incomplete Freund's adjuvant and other commercially available adjuvants.
- SPLPS sodium phthalyl lipopolysaccharide
- Freund's complete adjuvant/Incomplete Freund's adjuvant and other commercially available adjuvants.
- the present invention relates to a new vaccine, in which the DT or TT, as carriers, have been replaced by a set of peptides which can bring in the T helper cells without the risk of causing immuno-suppression of antibody production. Furthermore, the choice of these peptides has been made in a manner so as to evoke immune response by individuals of different genetic background. This innovation will enlarge the positivity of antibody response of the new vaccine and avoid the unwelcome suppression, which the use of the entire DT and TT toxoids as carriers bring in.
- LHRH or GnRH is common to both males and females! This is of advantage as the anti- LHRH vaccines designed for use in males will also have application in females.
- LHRH agonists alone or in combination have been used to block estrogen and progesterone thereby controlling the breast cancer in women (Spicer and Pike, 2000; Burger et al, 1996). With LHRH agonists combined with tamoxifen, Klijn et al. (2001) achieved immunocastration equivalent to surgical castration, in women with advanced breast cancer. This endocrine therapy yielded significant progression-free survival benefit (6.8 years) in clinical trials.
- LHRH is a conserved molecule; the sequence of the decapeptide is similar in humans and animals. Therefore, any LHRH based regimen designed for humans, will also have potential in affecting the LHRH controlled biological phenomena in animals.
- a monoclonal antibody generated against LHRH/GnRH was used to suppress heat/estrus of female dogs.
- Reversible control of fertility Recombinant anti-LHRH vaccines can also be used in providing reversible control of male fertility in addition to the management of prostate carcinoma.
- LHRH peptide was coupled to diphtheria toxoid (DT) and used to immunize rats at 4-week intervals. This method caused significant reduction in weights of reproductive organs without any side-effects.
- the histopathology revealed marked changes in the gonads and the accessory sex organs including the prostate. Spermatogenesis and fertility was restored after 300 days of vaccination along with below normal prostate recovery when the antibodies were no longer in circulation.
- anti-LHRH vaccines prepared by using oligonucleotide sequence of SEQ ID No. 1 and 2, and polypeptide sequence of SEQ ID No. 3 and 4, can be employed if required for reversible control of male fertility.
- Meat quality oligonucleotide sequence of SEQ ID No. 1 and 2, and polypeptide sequence of SEQ ID No. 3 and 4
- the present invention provides an embodiment of DNA fragments comprising SEQ. ID. No. 1 or SEQ. ID. No. 2 useful for the preparation of proteinic and DNA vaccines for the treatment of prostate cancer dependent on LHRH/GnRH and testosterone in mammals like humans.
- an embodiment of the invention features methods capable of providing a process for immunological castration without surgery.
- An embodiment of the invention provides methods capable of providing a process for reducing prostate size without surgery.
- An embodiment of the invention features methods capable of providing products and processes for the treatment of prostate cancer, dependent on sex-steroid hormone, testosterone, in men.
- An embodiment of the invention features methods capable of providing products and processes for the treatment of breast cancer, dependent on sex-steroid hormones, estrogen and progesterone, in women.
- An embodiment of the invention features methods also capable of providing products and processes for the reversible control of fertility and hormone dependent manifestations such as estrus/heat of female companion animals like dogs and cats.
- An embodiment of the invention features methods capable of providing products and processes for blocking the testosterone production to improve the quality of meat in animals raised for this purpose like pigs, rams, etc.
- Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
- prostate cancer or carcinoma of prostate refers to a disease pertaining to males in which prostate cells multiply at unusual rate resulting in enlargement of prostate gland or tumor formation.
- DNA vaccine is a composition, i.e., plasmid DNA which is free of the genes that are present in the genome of the bacterial host used to amplify, extract and purify the plasmid DNA.
- the term therefore includes a recombinant DNA incorporated into an autonomously replicating plasmid/vector. It also includes a separate molecule such as cDNA, a genomic fragment, a fragment produced by polymerase chain reaction, or a restriction fragment.
- the isolated DNA is substantially free of other cellular components of the bacterial host (e.g., free from protein components), or the culture medium when used to grow the bacteria.
- DNA vaccination means a method in which DNA containing the sequences encoding for an "antigenic determinant", is administered to the recipient. Such methods can result in the expression of the antigenic determinant in the recipients thereby eliciting an immune response.
- Proteinic vaccine refers to a composition, i.e., protein, which is free from other proteins that are present in the bacterial host used to extract and purify the protein.
- the term therefore includes a recombinant protein incorporated into a bacterial host through an autonomously replicating plasmid/vector.
- the isolated protein is substantially free of other cellular components of the bacterial host (e.g., free from DNA, RNA components), or the culture medium when used to grow the bacteria.
- protein vaccination means a method in which recombinant protein containing an "antigenic determinant", is administered to the recipient. Such methods can result in the expression of anti-antigenic determinant in the recipients thereby eliciting an immune response.
- an "antigenic determinant” refers to any agent, which can elicit an immunological response in an individual.
- the immunological response may be mediated by B-and/or T-lymphocytic cells.
- the "antigenic determinants” are generally used to refer to a part of protein molecule, which contains LHRH peptides linked with other T cell peptides. T-cell peptides are those features which induce a T-cell response.
- an "antibody” is used to imply immunological response, which is generated when an antigenic determinant is administered to a recipient.
- the antibody is generally specific to the "antigenic determinant” in terms of reactivity.
- an "endonuclease” refers to an enzyme found in bacteria which causes nicks in the DNA thus making it redundant for its biological utility.
- a "protease” refers to an enzyme found in bacteria which degrades proteins thus making it redundant for its biological utility.
- a "nucleotide sequence” refers to single- or double-stranded DNA and RNA sequences. The term describes molecules that include any of the known bases of DNA and RNA. The nucleotide sequence is transcribed (in the case of DNA) and translated (in the case of mRNA) into peptide or protein when 'operably linked' to appropriate regulatory sequences.
- Promoter, Intron and Polyadenylation sequences, ribosome binding site, along with other regulatory sequences, are required for the transcription and translation of the nucleotide sequence in the recipient cell.
- a "eukaryotic expression vector” is referred to circular DNA molecules, i.e plasmids, which can replicate autonomously inside an appropriate eukaryotic host. This can be used to carry desired nucleotide sequence " for its expression in the recipients.
- Promoter, intron, termination and polyadenylation sequences can also be present in the vector.
- operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
- control sequences operably linked to a coding sequences are capable of affecting the expression of the nucleotide sequence.
- an "adjuvant” is a compound or mixture of compounds, which enhances the ability of a vaccine to elicit an immune response.
- delivery system refers to mode of vaccine delivery.
- nucleotide sequence of SEQ. ID. No. 1 which is useful for the preparation of DNA and proteinic vaccines competent to generate anti-LHRH response after insertion into a eukaryotic vector and prokaryotic vector, respectively, and injection as per the procedures described in the text.
- nucleotide sequence of SEQ. ID. No. 1 which is useful for the preparation of DNA and proteinic vaccines competent to generate anti-LHRH response after insertion into a eukaryotic vector and prokaryotic vector, respectively, and injection as per the procedures described in the text.
- the present invention provides a method for eliciting an immune response against a selected antigenic determinant using DNA and protein vaccine techniques, in rats, which are studied as animal models. As humans and rats have the same antigenic determinant, the method proven in rats, can be extended to humans to provide a suitable therapeutic immune response in the treatment of prostate or breast cancers, which are sex steroid hormone-dependent tumors.
- Figure 1 shows the schematic construct of a typical though not unique recombinant plasmid, useful for the preparation of DNA vaccine.
- the plasmid contains a DNA fragment corresponding to nucleotide sequence of SEQ. ID. No. 1 or SEQ. ID. No. 2.
- Figure 2 shows the schematic construct of a typical though not unique recombinant plasmid, useful for the preparation of proteinic vaccine.
- the plasmid contains a DNA fragment corresponding to nucleotide sequence of SEQ. ID. No. 1 or SEQ. ID. No. 2.
- Figure 3 shows generation of antibodies against LHRH in rats injected with the DNA vaccine (A) containing DNA fragment of SEQ. ID. No.l (B) containing DNA fragment of SEQ. ID. No.2. Note that each of the eight animals has produced antibodies reactive with LHRH, and the antibody response is of long duration after two intramuscular injections of the DNA vaccine.
- Figure 4 shows the decline of testosterone levels in 4 different animals immunized with the plasmid DNA vaccine. ' Note that testosterone declines drastically in each animal after immunization.
- Figure 5 shows generation of antibodies against LHRH in rats injected with the proteinic . vaccine (A) containing DNA fragment of SEQ. ID. No.2 (B) containing DNA fragment of
- Figure 6 shows the decline of testosterone levels in 4 different animals immunized with the proteinic vaccine. Note that testosterone declines drastically in each animal with the emergence of antibodies.
- Figure 7 shows the histology of a representative extirpated prostate lobe in comparison to that of a normal prostate isolated from a rat, which was not immunized. It can be seen from the diagram that immunization causes shrinkage in prostate tissue mass.
- the nucleotide sequence of the DNA fragment selected in the present invention to encode for an antigenic determinant was designed in such a way that 5 repeat units of nucleotide sequence encoding LHRH peptide, were interspersed among the nucleotide sequences encoding 5 different small peptides recognizing determinants on the T type of lymphocytes, which play a crucial helper role in motivating the antibody generator B cells to produce antibodies.
- Two designs of the DNA fragment having sequence of SEQ. ID. No. 1 and SEQ. ID. No. 2, encoding for antigenic determinant (i.e. multimeric LHRH peptides) used in the present invention Two designs of the DNA fragment having sequence of SEQ. ID. No. 1 and SEQ. ID. No. 2, encoding for antigenic determinant (i.e. multimeric LHRH peptides) used in the present invention.
- the nucleotide sequence corresponding to 2 designs differed in the placement of T- cell peptides amongst the repeat units of LHRH peptide.
- DNA fragments corresponding to the designed nucleotide sequences were synthesized by using the known techniques of genetic engineering in the art, like joining short fragments resulting from custom synthesized over-hanging nucleotides followed by filling in by polymerase chain reaction technique.
- the DNA fragments have unique EcoR I and Sal I restriction enzyme sites in the beginning and end, respectively, to facilitate insertion of these fragments into an appropriate expression vector.
- the synthesized DNA fragments conforming to the above designs were inserted into the eukaryotic expression or prokaryotic expression vectors by the standard recombinant DNA techniques known in the art.
- a number of eukaryotic vectors are available for making DNA vaccine, from commercial and academic sources.
- the regulatory/control sequences will depend on the host being treated and the type of preparation used. Thus, if the host's endogenous transcription and translation ⁇ machinery will be used to express the proteins, the control or regulatory sequences compatible with the particular host will be utilized.
- promoters for use in mammalian systems include, but are not limited to, promoters derived from SN40, CMN, HSN, RSV, MMTN, among others.
- the eukaryotic expression vector based on a strong cytomegalovirus (CMN) promoter was used.
- the vector also contained cytomegalovirus intron A and BGH derived polyadenylation sequences for regulating the expression.
- the D ⁇ A fragment was cloned into the expression vector by known techniques in the art like restriction digestion, ligation, etc.
- Fig. 1 shows a representative, though not unique, schematic construct of eukaryotic expression vector, which contains the cloned D ⁇ A fragment having nucleotide sequences as per SEQ. ID. ⁇ o.l or 2.
- the D ⁇ A conforming to the above vaccine design can be prepared in large amounts and with high fidelity and accuracy using a bacterial host, as described hereunder.
- E. coli Escherichia coli
- DHl Escherichia coli
- DH5 ⁇ Escherichia coli
- C600 Escherichia coli
- XLl-Blue Escherichia coli
- the present invention employed E. coli strain DH5 ⁇ for the extraction and purification of plasmid DNA, the detailed process of which comprising of:
- the ingredients mentioned in the said process can be varied in the range of 10% for obtaining similar level of purity and yield of the plasmid DNA.
- the purity-check and quantifications of isolated plasmid DNA can be done by agarose gel- electrophoresis and spectrophotometeric absorption.
- the ratios of the absorbance at 260 nm and 280 nm wavelength, of the DNA samples, were observed in the range of 1.8-1.9, which is a standard for a good plasmid DNA preparation.
- prokaryotic vectors are available for making recombinant proteins, from commercial and academic sources.
- the regulatory/control sequences will depend on the host being treated and the type of preparation used. Thus, if the host's endogenous transcription and translation machinery will be used to express the proteins, the control or regulatory sequences compatible with the particular host will be utilized.
- promoters for use in bacterial systems include, but are not limited to; T7, T5, tac, lac, pH, ⁇ P , among others.
- the prokaryotic expression vector based on a strong T7 promoter, was used.
- the DNA fragment was cloned into the expression vector by known techniques in the art like restriction digestion, ligation, etc.
- Fig. 2 shows a representative, though not unique, schematic construct of prokaryotic expression vector, which contains the cloned DNA fragment having nucleotide sequences as per SEQ. ID. No. l or 2.
- the recombinant protein conforming to the above vaccine design can be prepared in large amounts and with high fidelity and accuracy using a bacterial host, as described hereunder.
- a large number of bacterial hosts with low or no protease activity are known in the art for extraction and purification of recombinant proteins. These include, but not limited to, ' Escherichia coli strains BL21, BL21(DE3), BL21(DE3)pLys, TGI, XLl-Blue.
- the present invention employed E. coli strain BL21(DE3) for the extraction and purification of plasmid .
- DNA the detailed process of which comprising of:
- washing buffer A 100 mM NaH 2 PO , 500 mM NaCl, 10 mM Tris, 6 M Urea, pH 6.5
- washing buffer B 100 mM NaH 2 PO 4 , 500 mM NaCl, 10 mM Tris, 6 M Urea, pH 5.9.
- elution buffer 100 mM NaH 2 PO 4 , 500 mM NaCl, 10 mM Tris, 2 M Urea, pH 4.2.
- the plasmid DNA can be administered as vaccine, alone or along with a compatible adjuvant.
- DNA encapsulated Microparticles of poly-lactic-co-glycolic acid (PLGA) copolymer can also be used for injection.
- the recipients can be injected with DNA vaccine through any parenteral route, e.g., intramuscular, intradermal, subcutaneous, intranasal, oral routes.
- Muscle is a useful tissue for the delivery and expression of plasmid-coded genes because mammals have a proportionately large muscle mass, which can be readily accessed by direct injection through skin. A comparatively large dose of plasmid DNA can be deposited into muscle by multiple and/or repetitive injections. Multiple injections can also be used for therapy over extended periods of time.
- Present invention involved free plasmid DNA injection without any carrier. It was injected intramuscularly and it resulted in eliciting an immune response. Two or more injections at a dose of 100 ⁇ g each, given at about 15 days interval, were sufficient to generate anti-LHRH antibodies. The dose amount can be varied from 20 ⁇ g to 500 ⁇ g, each generating equal and sufficient response, causing a decline of testosterone to castration levels along with reduction in the prostate size.
- the said proteinic vaccine can be mixed with alum, sodium phthalyl lipopolysaccharide (SPLPS) or with other commercially available adjuvants. It can also be delivered as Microparticles of . poly lactide, poly glycolide or poly-lactic-co-glycolic acid (PLGA) copolymer, and Nano particles of calcium phosphate.
- SPLPS sodium phthalyl lipopolysaccharide
- PLGA poly-lactic-co-glycolic acid copolymer
- Nano particles of calcium phosphate for experimental purposes and with the idea of demonstrating the proof of concept in the ability of the invented sequences to cause reduction of testosterone to castration level and bring about atrophy of the prostate, the vaccine was given intramuscularly to rats with Freund's Complete Adjuvant, followed by a booster after 15 days with Freund's Incomplete Adjuvant. The dose amount was varied in the range 20-500 ⁇ g per animal. Each animal elicited immune response in terms of anti-LHRH antibodies
- DNA vaccine was injected intramuscularly twice at 30 days interval, to male adult rats at a dose of 100 ⁇ g.
- the efficacy of the vaccine was determined in terms anti-LHRH antibody production, which was quantified by ELISA technique using LHRH as antigen.
- the vaccine is competent to produce antibodies measurable in blood, which are reactive with LHRH.
- the antibody response was produced in all animals in which immunization was done, irrespective of the genetic background of the mice or rats. Fig.
- the DNA fragments with nucleotide sequences as described in the text were cloned into a prokaryotic expression vector.
- This recombinant expression vector was used to transform a bacterial host, and then recombinant protein isolated and purified for use as proteinic vaccine.
- Proteinic vaccine mixed with Freund's Complete Adjuvant (FCA) was injected intramuscularly, to male adult rats at a dose of 100 ⁇ g. Two booster doses were given along with Freund's Incomplete Adjuvant, at 15 days interval.
- FCA Freund's Complete Adjuvant
- the efficacy of the vaccine was determined in terms anti-LHRH antibody production, which was quantified by ELISA technique using LHRH as antigen.
- the vaccine is competent to produce antibodies circulating in blood, which are reactive with LHRH.
- Fig. 5 gives the results of antibody titres generated by proteinic vaccines containing DNA fragments having nucleotide sequences as per SEQ. ID. No. 1 and 2.
- Each one of the four animals in each group produced antibodies.
- the antibody titres in each rat were higher than those required for exercising biological effect, which is evidenced by the data on testosterone levels, which declined in tune with emergence of antibodies.
- Fig. 6 shows declining testosterone levels perceptively with the production of the antibodies.
- the biological effect of immunization on prostate size was determined by removing the prostate lobes surgically, the weights of which were significantly lower than those of control rats, which were either not immunized or immunized with FCA alone (Table 1). Further confirmation of the atrophy of the prostate was obtained by the histology of the extirpated prostate lobe as is evident from a representative figure (Fig.7).
- This invention provides the nucleotide sequence of DNA fragments comprising SEQ. ID. No. 1 or SEQ. ID. No. 2, and their insertion into a prokaryotic and a eukaryotic expression vector, for making a proteinic and DNA vaccine, respectively.
- the advantages associated with this invention comprise the following: (a) The invention provides an immunological process to reduce prostate size without surgery in patients suffering from benign prostate enlargement. It will be a convenient approach avoiding hospitalization costs particularly beneficial to those patients, in whom surgery is hazardous for other medical reasons.
- the invention provides products and processes for generating antibodies competent to react with native LHRH/GnRH to reduce testosterone levels to castration level without orchiectomy.
- the invention provides products and processes for the treatment of prostate cancer, dependent on LHRH/GnRH and testosterone in mammals like humans.
- the invention provides products and processes wherein the vaccines can be used in place of costly LHRH agonists and antagonists which are used in clinical practice to block the action of LHRH hormone in the therapy of carcinoma of prostate.
- the invention provides a method wherein the administration of the vaccine in the form of DNA, does not need any oil based adjuvant such as squalene,
- the invention provides products and processes wherein the vaccines can be used in other sex-steroid hormone dependent diseases like breast cancer in women.
- the invention provides products and processes wherein the vaccines can be used for reversible control of fertility and hormone dependent manifestations such as estrus of female companion animals like dogs and cats.
- the invention provides products and processes wherein the vaccines can be used to block the testosterone production to improve the quality of meat in
Abstract
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PCT/IN2004/000153 WO2004105791A2 (fr) | 2003-06-02 | 2004-06-02 | Vaccins anti-lhrh de recombinaison |
Country Status (1)
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WO (1) | WO2004105791A2 (fr) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
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US5759551A (en) * | 1993-04-27 | 1998-06-02 | United Biomedical, Inc. | Immunogenic LHRH peptide constructs and synthetic universal immune stimulators for vaccines |
US5869103A (en) * | 1994-06-18 | 1999-02-09 | Danbiosyst Uk Limited | Polymer microparticles for drug delivery |
EP1035133A2 (fr) * | 1999-02-17 | 2000-09-13 | Pfizer Products Inc. | Protéines de fusion comprenant des supports capables d' induire une double réponse immunitaire |
US6521746B1 (en) * | 1991-10-16 | 2003-02-18 | University Of Saskatchewan | Polynucleotides encoding LKT 111 |
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2004
- 2004-06-02 WO PCT/IN2004/000153 patent/WO2004105791A2/fr active Application Filing
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6521746B1 (en) * | 1991-10-16 | 2003-02-18 | University Of Saskatchewan | Polynucleotides encoding LKT 111 |
US5759551A (en) * | 1993-04-27 | 1998-06-02 | United Biomedical, Inc. | Immunogenic LHRH peptide constructs and synthetic universal immune stimulators for vaccines |
US5869103A (en) * | 1994-06-18 | 1999-02-09 | Danbiosyst Uk Limited | Polymer microparticles for drug delivery |
EP1035133A2 (fr) * | 1999-02-17 | 2000-09-13 | Pfizer Products Inc. | Protéines de fusion comprenant des supports capables d' induire une double réponse immunitaire |
Non-Patent Citations (4)
Title |
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FUERST JOHANNES ET AL: "Effect of active immunization against luteinizing hormone-releasing hormone on the androgen-sensitive Dunning R3327-PAP and androgen-independent Dunning R3327-AT2.1 prostate cancer sublines" PROSTATE, vol. 32, no. 2, 1997, pages 77-84, XP002315521 ISSN: 0270-4137 cited in the application * |
GUPTA JAGDLSH C ET AL: "Engineering, cloning, and expression of genes encoding the multimeric luteinizing-hormone-releasing hormone linked to T cell determinants in Escherichia coli" PROTEIN EXPRESSION AND PURIFICATION, vol. 37, no. 1, September 2004 (2004-09), pages 1-7, XP004523931 ISSN: 1046-5928 * |
RAINA KORNAL ET AL: "Purification, refolding, and characterization of recombinant LHRH-T multimer" PROTEIN EXPRESSION AND PURIFICATION, vol. 37, no. 1, September 2004 (2004-09), pages 8-17, XP004523932 ISSN: 1046-5928 * |
TALWAR G P: "Fertility regulating and immunotherapeutic vaccines reaching human trials stage" HUMAN REPRODUCTION UPDATE, vol. 3, no. 4, July 1997 (1997-07), pages 301-310, XP002315522 ISSN: 1355-4786 * |
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WO2004105791A3 (fr) | 2005-04-07 |
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